In the STAT system of proteins, we have determined the solution structure of the N-terminal domain of STAT4, utilizing new NMR methods, and found evidence for a new dimerization interface. Corroborating evidence for this interface is being pursued via investigation of the N-terminal domains (NTDs) of three other STAT family members. Our studies involve both NMR and biophysical methods. The interactions indicated in the NTD studies have also enabled us to examine the heterodimerization of STAT1 and STAT2 NTDs. These studies are key to understanding the variety of modes of combination among the STAT proteins that can explain the involvement of the seven STAT proteins in literally hundreds of signaling/DNA recognition processes. The studies are being supplemented with further structural determinations for other STAT NTDs. In this reporting year, we have been worked on new methods to tag the proteins with paramagnetic centers and obtain valuable distance and orientational data. Tagging through single-cysteine mutations has been developed, and optimization of the type of tag to utilize continues. Experimentation with new lanthanide-binding tags (see Methods Development project) proceeds to select the appropriate system. The tagging approach also is designed to allow us to utilize pulsed EPR methods (DEER) that are available via a trans-NIH project involving NCI, NIDDK, and NHLBI. Initial DEER data are encouraging and will be extended. We have also collected small-angle X-ray scattering (SAXS) data with the assistance of Dr. Yun-Xing Wang to facilitate these studies of the STAT NTD homo- and hetero-dimers. The controversial nature of these structures requires that multiple complementary tools be used to validate the structural findings.